The differentiation antigens have emerged as targets for cancer therapy. We will investigate DNA vaccines that are designed to break tolerance and enhance potency of immunization against melanoma differentiation Melanoma differentiation antigens will be targeted, including tyrosine, gp75TRP-1, TRP-2 and gp100. These antigens cover early and late stages of melanocyte/melanoma differentiation and are specifically selected to overcome tumor heterogeneity. These differentiation antigens are recognized by antibodies and both CD4+ and CD4+ T cells of patients with melanoma. This raises the possibility that vaccines can be designed that general humoral and cellular responses, including helper and cytotoxic T cells. Our results in laboratory models show that both antibody and T cells responses can be induced against tumor differentiation antigens by DNA immunization, but genes encoding altered antigens are required. for effective immunity. Because differentiation antigens are self antigens that are likely to be weakly immunogenic, we will investigate and compare three approaches of DNA vaccination to enhance immunogenicity and/or overcoming immune tolerance: 1. Immunization with xenogeneic DNA; 2. Immunization with DNA encoding antigens that sort to antigen presenting compartments; 3. Immunization with DNA minigenes encoding heteroclitic peptides, and 4. Augmentation of immunity by GM-CSF DNA. We expect that these approaches will lead to development of DNA vaccine of antigens of melanoma.